The enzymes of galactosyltransferase family exhibit diverse functions, namely: they are involved in sugar transfer, cell-cell adhesion and pattern formation during development. The sugar acceptor and donor specificity of some of the enzymes can be modulated by alpha-lactalbumin (LA), a mammary gland specific calcium binding protein that has sequence and structural similarity to the c- type lysozymes (1). Since the enzymes of galactosyltransferase family are multifunctional, we are currently investigating the structure and function of various regions of the beta-1,4 galactosyltransferase(beta-1,4 GT) and its interactions with LA to account for the diverse functions. Currently, using the genetic engineering and crystal structure determination methods, the aim of our laboratory is to determine the three-dimensional structure of the catalytic domain, full length protein containing transmembrane (TM) domain and the complex beta-1,4 GT and its substrate modifier protein LA. The studies are aimed to define the sugar nucleotide and sugar acceptor binding specificities of the catalytic domain of beta-1,4 GT family, identify the region(s) that interact with LA in the presence of sugar which change the sugar acceptor specificity of the enzyme. Furthermore, our aim is to characterize the region that is involved in cell adhesion and interactions with laminin and other cell-matrix proteins. This structural information will be beneficial in understanding the role of glycosyltransferases at the cell surface. Earlier in our laboratory, the functional analysis of the TM domain was carried out by expressing and localizing the TM mutants in mammalian cells and some of the structural requirements of the TM domain were correlated with the function (2). For the structure and function analysis of the stem region, the sugar donor and acceptor binding regions of the catalytic domain, and for the studies on sugar-dependent protein-protein interactions between beta-1,4 GT and LA the recombinant proteins have been expressed in E.coli, proteins renatured from the inclusion bodies, folded in vitro, and analyzed for substrate bindings and enzymatic activities (3). These results have shown that the catalytic domain lies at the COOH-terminal portion of beta-1,4 GT and can be further subdivided into the N[unreadable] and C-terminal halves. The major binding region for the sugar acceptor lies in the N-terminal half of the catalytic domain, while the binding of the sugar nucleotide donor is localized to the C-terminal half, and in order that the catalysis to occur the two halves have to overlap at the catalytic surface. The O4-H of the GlcNAc has to be in the close proximity of the C1 of galactose (UDP-alpha-galactose) at the catalytic site, and the inversion of the configuration at C1 has to occur to generate a beta-glycosidic linkage. The disulfide bond between Cys 134 and Cys 247 is required during catalysis, but not for folding or binding substrates. The recombinant beta-1,4 GT and rat LAs, the short and long forms, and in vivo produced rat LAs have been crystalized for structure determination. The structure determination of some of these proteins are in progress.